The present invention relates to non-naturally occurring nucleic acid and peptides corresponding to nucleic acid and peptides of Mycobacterium tuberculosis and Mycobacterium leprae. The nucleic acid and peptides of the present invention have utility for diagnostics and therapeutics.
Mycobacterium tuberculosis is the causative agent of tuberculosis. Tuberculosis is a chronic bacterial infection characterized by the formation of granulomas in infected tissues and by cell mediated hypersensitivity. The usual site of the disease is the lungs but other organs may be involved. In countries where human immuno-deficiency virus (HIV) infection is endemic, tuberculosis is a frequent cause of morbidity in AIDS patients. Tuberculosis has shown a resurgence in recent years worldwide. Mycobacteria contain an array of protein and polysaccharide antigens giving rise to a cell mediated hypersensitivity. The hypersensitivity is often used to diagnose tuberculosis and to monitor the disease pathogenesis. See: Harrison""s Principles of Internal Medicine, Twelfth Edition, McGraw-Hill, Inc., (1991), pp. 637-645.
Mycobacterium leprae is the causative agent of Hanson""s disease or leprosy. Leprosy is a chronic infection of superficial tissues, especially the skin and peripheral nerves. Mycobacterium leprae multiplies slowly, and has not been grown in tissue culture or artificial media. Mycobacterium leprae does not elicit strong immunological responses in infected individuals. However, a serodiagnostic test for the detection of antibody to Mycobacterium leprae antigen is used to aid in the diagnosis. See Harrison, supra, pp. 645-648.
Patient care, as well as the prevention and transmission of Mycobacterium tuberculosis and leprae, requires reliable diagnostic and prognostic tools to detect nucleic acid, antigens and antibodies relating to tuberculosis and leprosy.
A number of therapeutic agents are currently available for combatting Mycobacterium tuberculosis and Mycobacterium leprae infections in man. However, limitations to these therapies, particularly for Mycobacterium tuberculosis infection, demonstrate that new, more effective agents are needed. For example, agents such as isoniazid and rifampicin are currently in use for Mycobacterium tuberculosis infections, but strains which are resistant to one or the other or both of these agents are being reported with increasing frequency (eg., Chawla et al., 1992, Am. Rev. Respir. Dis. 146, 278-279). In addition, Mycobacterium tuberculosis bacilli treated by drug therapy frequently remain viable, but dormant as latent infections in macrophage phagolysosomes which may re-emerge as full-blown tuberculosis at a later date (eg., Dannenberg, in xe2x80x9cThe Mycobacteria: A Sourcebookxe2x80x9d, Kubica and Wayne, eds., Dekker, N.Y., pp. 721-760).
This invention relates to diagnostics and therapeutics for Mycobacterium tuberculosis, M. leprae and other mycobacterial species including, but not limited to: M. avium, M. bovis, M. chitae, M. fortuitum, M. gorodonae, M. intracellulare, M. kansaii, M. paratuberculosis, M. smegmatis, M. terrae, and M. ulcerans. One embodiment of the present invention features, as an article of manufacture or as a composition, a non-naturally occurring nucleic acid having twenty or more nucleotides in a sequence corresponding to a sequence of Mycobacterium tuberculosis or Mycobacterium leprae nucleic acid. The non-naturally occurring nucleic acid of the present invention is exemplified by Seq. ID No. 1 with respect to Mycobacterium tuberculosis and by Seq. ID Nos. 23-26 and 120-140 with respect to Mycobacterium leprae. Preferably, the sequence has at least twenty, and more preferably at least thirty nucleotides in a sequence corresponding to Mycobacterium tuberculosis or Mycobacterium leprae nucleic acid. The sequence may correspond to the entire coding sequence for the gene or a part of the coding sequence. However, sequences larger than 1000 nucleotides in length are difficult to synthesize.
One embodiment of the present invention features a non-naturally occurring nucleic acid wherein the nucleic acid encodes a peptide of Mycobacterium tuberculosis or M. leprae or other mycobacterial species. The peptide has utility for diagnostics and therapeutics.
A further embodiment of the present invention comprises a non-naturally occurring nucleic acid wherein the nucleic acid is capable of binding messenger RNA of Mycobacterium tuberculosis or M. leprae or other mycobacterial species. Such non-naturally occurring nucleic acid is capable of acting as anti-sense nucleic acid to control the translation of messenger RNA of Mycobacterium tuberculosis, M. leprae or other mycobacterial species. The non-naturally occurring nucleic acid inhibits the translation of Mycobacterium tuberculosis, M. leprae or other mycobacterial gene products, which by way of example, relate to the synthesis of antibiotics and the constituents of the cell wall, intermediary metabolism, transport processes, nucleic acid biosynthesis and modification, and antibiotic resistance. One of ordinary skill in the art would readily be able to discern which proteins were involved in the above metabolic processes. The above listed processes, as well as the proteins involved in such processes, are well described in any cellular biology textbook such as Molecular Cell Biology, Darnell, J., Lodish, H., and Baltimore, D. Scientific American Books, N.Y. Such non-naturally occurring nucleic acids have utility in therapeutics and diagnostics. A further embodiment of the present invention features a non-naturally occurring nucleic acid which nucleic acid is capable of binding specifically to Mycobacterium tuberculosis or leprae nucleic acid. Such non-naturally occurring nucleic acid has utility as probes and as capture reagents.
One embodiment of the present invention features an expression system comprising an open reading frame of DNA corresponding to Mycobacterium tuberculosis or leprae DNA. The nucleic acid further comprises a control sequence compatible with an intended host. The expression system is useful for making peptides corresponding to Mycobacterium tuberculosis or leprae nucleic acid.
A further embodiment of the present invention features a cell transformed with the expression system to make Mycobacterium tuberculosis or leprae proteins and peptides.
Several non-naturally occurring nucleic acids of the present invention feature nucleic acid relating to products involved in cell wall biosynthesis of Mycobacterium tuberculosis. Such nucleic acids have sequences of twenty or more nucleotides which correspond to sequences of Seq. ID No. 1. One such nucleic acid has a sequence of twenty or more nucleotides which correspond to a sequence within nucleotides 28,325 to 31,285 of Seq. ID No. 1. Nucleotides 28,325 to 31,285 encode a gene for polyketide or fatty acid synthesis. The gene codes for an enzyme with acyl transferase, enoyl reductase and dehydratase domains. The putative amino acid sequence of the gene product is set forth in Seq. ID No. 2.
An enzyme, ketoacyl ACP synthase of Mycobacterium tuberculosis, is encoded by nucleotides 26750 to 28237 of Seq. ID No. 1. The putative amino acid sequence of the enzyme is set forth in Seq. ID No. 3.
Nucleic acid encoding a gene for the enzyme beta-keto reductase of Mycobacterium tuberculosis corresponds to nucleotides 24636 to 26753 of Seq. ID No. 1. The putative amino acid sequence is set forth in Seq. ID No. 4.
Nucleic acid encoding COA ligases correspond to nucleotides 22136 to 23371 or 23251 to 23994 of Seq. ID No. 1. The putative amino acid sequence is set forth in Seq. ID No. 5 and Seq. ID No. 118.
Nucleic acids encoding two UDP-sugar transferases of Mycobacterium tuberculosis corresponds to nucleotides 9489 to 10846 of Seq. ID No. 1 and 12604 to 13995 of Seq. ID No. 1. The putative amino acid sequence are set forth in Seq. ID Nos. 6 and 7.
Nucleic acid encoding a methyltransferase of Mycobacterium tuberculosis corresponds to nucleotides 20006 to 19347 of Seq. ID No. 1. The putative amino acid sequence is set forth in Seq. ID No. 8.
Nucleic acid encoding a KdtB protein of Mycobacterium tuberculosis corresponds to nucleotides 5790 to 6275 of Seq. ID No. 1. KdtB protein is an essential 12 kD protein associated with the synthesis of lipopolysaccharide. The putative amino acid sequence is set forth in Seq. ID No. 9.
A further non-naturally occurring nucleic acid of the present invention has a sequence of twenty nucleotides which correspond to proteins involved in intermediary metabolism. One non-naturally occurring nucleic acid corresponds to a pyruvate carboxylase of Mycobacterium tuberculosis. Pyruvate carboxylase is involved in gluconeogenesis. The enzyme catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate in the presence of cofactors biotin and zinc. The nucleotides 1565 to 4939 of Seq. ID No. 1 encode the enzyme pyruvate carboxylase. The putative amino acid sequence of the enzyme is set forth in Seq. ID No. 10.
One non-naturally occurring nucleic acid has a sequence of twenty or more nucleotides corresponding to a phosphoribosylglycinamide formyltransferase. Phosphoribosylglycinamide formyltransferase catalyzes the third step in de novo purine biosynthesis. The nucleotides encoding phosphoribosylglycinamide formyltransferase are 8061 to 7144 of Seq. ID No. 1. The putative amino acid sequence of the enzyme is set forth in Seq. ID No. 11.
A further non-naturally occurring nucleic acid of the present invention corresponds to gene products involved in transport processes. One non-naturally occurring nucleic acid encodes a structural gene for an anion pump protein of Mycobacterium tuberculosis. The nucleic acid has a sequence which corresponds to a sequence within nucleotides 9369 to 8149 of Seq. ID No. 1 which encode the pump. The putative amino acid sequence is set forth in Seq. ID No. 12.
A further non-naturally occurring nucleic acid has a sequence of twenty or more nucleotides which correspond to gene products involved in DNA biosynthesis and cell division. One non-naturally occurring nucleic acid of the present invention corresponds to a Mycobacterium tuberculosis cell division protein. A Mycobacterium tuberculosis cell division protein is encoded at nucleotide positions 5042 to 5704 of Seq. ID No. 1. The putative amino acid sequence is set forth in Seq. ID No. 13.
One embodiment of the present invention features a non-naturally occurring nucleic acid having a sequence of twenty or more nucleotides which correspond to gene products that are involved in antibiotic resistance. One non-naturally occurring nucleic acid of the present invention corresponds to a ribosomal RNA methylase. The nucleic acid has a sequence of twenty or more nucleotides which corresponds to a sequence within nucleotides 17223 to 17933 of Seq. ID No. 1 encoding for the enzyme. The putative amino acid sequence is set forth in Seq. ID No. 14.
A further non-naturally occurring nucleic acid has a sequence of twenty or more nucleotides which correspond to a sequence of Mycobacterium tuberculosis useful as a probe to identify Mycobacterium tuberculosis genes or homologous genes in other mycobacteria or other related bacterial species. Nucleic acid sequences that are specific to Mycobacterium tuberculosis genes correspond to nucleotides 15841 to 15203, nucleotides 15131 to 14306, nucleotides 20491 to 21489, nucleotides 911 to 1540, nucleotides 16223 to 17161, nucleotides 24020 to 24619, nucleotides 3 to 902, nucleotides 11313 to 11651, and nucleotides 11766 to 12503. The putative amino acid sequences are set forth in Seq. ID Nos. 15, 16, 17, 18, 19, 20, 21, 22, 118 and 119. These amino acid sequences may define proteins which are specific to Mycobacterium tuberculosis. Such proteins may give rise to antibodies which are specific to Mycobacterium tuberculosis. 
The Mycobacterium tuberculosis peptides, the functions of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 1 are summarized in Table I below.
Tables I-XXVI contain information on the genes encoded by the cosmid sequences with the corresponding Seq. ID Nos. (see table headings). Tables I-XIX consist of four columns with the heading gene, position, enzyme name or activity with Seq. ID No., and function. Tables XX-XXVI consist of five columns with the heading gene, SegID, position, enzyme or protein name, and function. Under the heading xe2x80x9cgenexe2x80x9d, a specific name is provided where the gene is positively identified by homology to other organisms, or a letter followed by the cosmid number. The letter xe2x80x9cdxe2x80x9d represents dehydrogenase, xe2x80x9ctxe2x80x9d represents transport associated, xe2x80x9caxe2x80x9d represents ATPase. Under the heading xe2x80x9cSeqIDxe2x80x9d, a specific sequence ID number is provided. Under the heading xe2x80x9cpositionxe2x80x9d, a range of nucleotides with the first and last numbers corresponding to the start and stop positions are identified which correspond to the respective Seq. ID No. In tables I-V, the start positions correspond to probable translation initiation codons; in tables VI-XXVI, the start positions correspond to the beginning of the reading frame which includes the predictable in vivo start site but usually is some distance away. Under the heading xe2x80x9cEnzyme name or activityxe2x80x9d, an exact name may be given, an activity (e.g., dehydrogenase), an indication of homology to other sequences in public databases, or the designationxe2x80x94M. leprae gene sequence, indicating a previously un-described gene. Under the heading xe2x80x9cfunctionxe2x80x9d, a number of functional categories, as described in the text are identified; for example, xe2x80x9cmetabxe2x80x9d denotes intermediary metabolism. In some cases metabolism is more specifically broken down in the following manner; xe2x80x9csynthxe2x80x9d denotes biosynthesis, xe2x80x9ccatabxe2x80x9d denotes breakdown. Further information is sometimes given on the compounds involved; xe2x80x9clipidxe2x80x9d denotes lipid, xe2x80x9caaxe2x80x9d denotes amino acid, xe2x80x9cntxe2x80x9d denotes nucleotide, xe2x80x9ccarboxe2x80x9d denotes carbohydrate, xe2x80x9cglycoxe2x80x9d denotes glycolysis, xe2x80x9cantibioticxe2x80x9d denotes antibiotics, xe2x80x9cproteinxe2x80x9d denotes xe2x80x9cproteinxe2x80x9d, xe2x80x9ctcaxe2x80x9d denotes tricarboxylic acid cycle, xe2x80x9cwallxe2x80x9d denotes cell wall, xe2x80x9ccofactxe2x80x9d denotes cofactor (an indication of the specific cofactor sometimes follows), xe2x80x9cM. tb-specificxe2x80x9d denotes an M. tuberculosis gene useful as a probe, xe2x80x9cM. leprae-specificxe2x80x9d denotes an M. leprae gene useful as a probe, xe2x80x9ctransportxe2x80x9d denotes a gene involved in transport, cell division denotes a gene involved in xe2x80x9ccell divisionxe2x80x9d, xe2x80x9cresistancexe2x80x9d denotes a gene implicated in antibiotic resistance, xe2x80x9crepairxe2x80x9d denotes a DNA repair gene, xe2x80x9ccell div.xe2x80x9d denotes a gene involved in cell division, xe2x80x9cantigenxe2x80x9d denotes a gene that is known to be a mycobacterial antigen, or an antigen in another organism that may be useful for vaccine development, xe2x80x9credoxxe2x80x9d denotes involved in electron transport, xe2x80x9cstressxe2x80x9d denotes a gene that is involved in cellular stress responses, xe2x80x9cregulatoryxe2x80x9d denotes regulatory gene, xe2x80x9ctranslationxe2x80x9d denotes a gene whose product is involved in ribosome function, and xe2x80x9crecombinationxe2x80x9d denotes a gene involved in recombination.
Several non-naturally occurring nucleic acids of the present invention feature nucleic acid relating to products of Mycobacterium Leprae. One non-naturally occurring nucleic acid of the present invention has a sequence of twenty or more nucleotides which correspond to a sequence within Seq.
ID Nos. 23-26 and 120-140. The Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 23 are summarized in Table II below.
Nucleotides 32443 to 32093 of Seq. ID No. 23 encode a hypothetical 12 kD protein in the DNAX-RECR intergenic region. The putative amino acid sequence is set forth in Seq. ID No. 27.
One non-naturally occurring nucleic acid of the present invention corresponds to a sequence encoding the enzyme aspartate-semialdehyde dehydrogenase. The enzyme, aspartate-semialdehyde dehydrogenase, is involved in intermediary metabolism and is encoded by nucleotides 23517 to 22465 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 28.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the enzyme glycerol kinase. The enzyme, glycerol kinase, is involved in intermediary metabolism. The nucleotides encoding glycerol kinase are 14121 to 12658 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 29.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the enzyme aspartate kinase. The enzyme, aspartate kinase, is involved in intermediary metabolism. The nucleotide sequence encoding aspartate kinase are 24783 to 23520 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 30.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the RecM protein. This protein is involved in DNA repair DNA replication and cell division. The nucleotides encoding RecM protein are 32078 to 31470 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 31.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds a sequence encoding to 2-isopropyl malate synthase. The enzyme, 2-isopropyl malate synthase, is involved in intermediary metabolism. The nucleotides encoding 2-isopropyl malate synthase are 25583 to 27298 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 32.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a penicillin binding protein, involved in antibiotic resistance. The nucleotides encoding the penicillin binding protein are 3032 to 5494 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 33.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding an anion-transporting ATPase involved in transport processes. The nucleotides encoding this transport protein are 1060 to 1914 of Seq. ID No. 23. The putative amino acid sequence is set forth in Seq. ID No. 34.
Non-naturally occurring nucleic acids having a sequence of twenty or more nucleotides which correspond to a sequence of Mycobacterium leprae are useful as probes to identify Mycobacterium leprae genes or homologous genes in other mycobacteria or other related bacterial species. Nucleic acid sequences that are specific to Mycobacterium leprae genes correspond to nucleotides 38 to 1063, nucleotides 1060 to 1917, nucleotides 1911 to 2204, nucleotides 5513 to 6622, nucleotides 7161 to 7474, nucleotides 10765 to 11355, nucleotides 11789 to 12388, nucleotides 14034 to 14276, nucleotides 14269 to 14880, nucleotides 15548 to 15922, nucleotides 28664 to 29812, nucleotides 29805 to 30512, nucleotides 33851 to 35230, nucleotides 35227 to 36546, nucleotides 18608 to 17988, nucleotides 22453 to 21305, nucleotides 33762 to 33325 of Seq. ID No. 23. The respective putative amino acid sequences are set forth in Seq. ID Nos. 35-51.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptide within Seq. ID No. 24 are summarized in Table III set forth below.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a CoA ligase modifying enzyme. The enzyme, CoA ligase, is involved in intermediary metabolism. The nucleotides encoding this CoA ligase are 16941 to 18311 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 52.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding dihydropteroate synthase (DHPS). The enzyme, dihydropteroate synthase is involved in intermediary metabolism. The nucleotides encoding DHPS are 28531 to 29322 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 53.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding DNA-3-methyladenine glycosidase I. The enzyme, DNA-3-methyladenine glycosidase I, is involved in intermediary metabolism. The nucleotides encoding this glycosidase are 30740 to 31315 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 54.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding glucose-1-phosphate adenyltransferase. The enzyme, glucose-1-phosphate adenyltransferase, is involved in intermediary metabolism. The nucleotides encoding this enzyme are 33059 to 34315 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 55.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a 68 kD thiamine pyrophosphate-requiring protein. The 68 kD enzyme is involved in intermediary metabolism. The nucleotides encoding this protein are 693 to 2900 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 56.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a probable antigen identified in Mycobacterium tuberculosis. The nucleotides encoding this probable antigen are 20486 to 21097 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 57.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding uroporphyrinogen decarboxylase. The enzyme, uroporphyrinogen decarboxylase, is involved in intermediary metabolism. The nucleotides encoding this enzyme are 7954 to 9044 of Seq. ID No. 24. The putative amino acid sequence is set forth in Seq. ID No. 58.
Non-naturally occurring nucleic acids having a sequence of twenty or more nucleotides which correspond to a sequence of Mycobacterium leprae are useful as a probe to identify Mycobacterium leprae genes or homologous genes in other mycobacteria or other related bacterial species. Nucleic acid sequences that are specific to Mycobacterium leprae genes include nucleotides at positions 4637 to 5104, nucleotides 25695 to 26399, nucleotides 26195 to 26758, nucleotides 27995 to 28217, nucleotides 29399 to 30484, nucleotides 30396 to 30740, nucleotides 30737 to 31315, nucleotides 31589 to 31756, nucleotides 34389 to 35042, nucleotides 6290 to 5001, nucleotides 6882 to 6292, nucleotides 9041 to 10396, nucleotides 19930 to 18315, nucleotides 23399 to 22446, nucleotides 32508 to 32260, nucleotides 10402 to 11097, nucleotides 35946 to 35617, nucleotides 12935 to 13234, nucleotides 13486 to 13779, nucleotides 21186 to 21488, nucleotides 21540 to 21827, nucleotides 22002 to 22355, and nucleotides 23315 to 24517 of Seq. ID No. 24. The putative amino acid sequences are set forth in Seq. ID Nos. 59-81.
Mycobacterium leprae peptides, the function of such peptides, the nucleotide positions associated with, such peptides within Seq. ID No. 25 are summarized in Table IV set forth below.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding alcohol dehydrogenase (ADH). The enzyme, alcohol dehydrogenase, is involved in intermediary metabolism and detoxification. The nucleotides encoding ADH are 3301 to 4332 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 82.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the Mycobacterium leprae 85-B antigen (alpha antigen). The nucleotides associated with 85-B antigen are 26808 to 25829 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 83.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a major mycobacterial antigen and the enzyme alkyl hydroperoxide reductase (AHPC). The enzyme, mycobacterial antigen, is a detoxifying enzyme. The nucleotides encoding AHPC are 12402 to 11818 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 84.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding bacterioferritin (BFR). Bacterioferritin is involved in iron detoxification and storage. The nucleotides encoding BFR are 15629 to 16105 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 85.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding alcohol dehydrogenase-T (ADH-T). The enzyme, alcohol dehydrogenase-T, is involved in intermediary metabolism. The nucleotides encoding ADH-T are 28957 to 28664 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 86.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a molybdenum or sulfate transport protein. The nucleotides encoding this transport protein are located at positions 139 to 396 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 87.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a cytochrome P450-like hydroxylase. This enzyme is involved in detoxification processes or antibiotic synthesis. The nucleotides encoding this protein are 22311 to 22078 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 88.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a hydrogen peroxide inducible regulatory protein (OXYR). The nucleotides OXYR are 12485 to 13447 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 89.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding an enzyme (S)-2-hydroxy-acid oxidase (GOX). The enzyme, GOX is involved in intermediary metabolism. The nucleotides encoding GOX are 9872 to 8631 of Seq. ID No. 25. The putative amino acid sequence is set forth in Seq. ID No. 290.
Non-naturally occurring nucleic acids having a sequence of twenty or more nucleotides which correspond to a sequence of Mycobacterium leprae are useful as probes to identify Mycobacterium leprae genes or homologous genes in other mycobacteria or other related bacterial species. Nucleic acid sequences that are specific to Mycobacterium leprae genes include nucleotides at positions 139 to 396, nucleotides 24755 to 24309, nucleotides 34108 to 33197, nucleotides 717 to 859, nucleotides 1047 to 1268, nucleotides 1928 to 2791, nucleotides 2951 to 3250, nucleotides 10854 to 11063, nucleotides 5683 to 4985, nucleotides 10433 to 10176, and nucleotides 24266 to 23821 of Seq. ID No. 25. The putative amino acid sequences are set forth in Seq. ID Nos. 91-101.
Mycobacterium leprae peptides, the function of such peptides and the sequences associated with such peptides within Seq. ID No. 26 are summarized in Table V set forth below.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a cold-shock protein (CSPA). The protein, CSPA, is involved in cellular regulation processes. The nucleotides encoding CSPA are 27760 to 28176 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 102.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a DNA helicase similar to a human DNA excision repair protein. The enzyme is involved in DNA synthesis and replication. The nucleotides encoding this helicase are 20362 to 19211 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 103.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the M. leprae fatty acid oxidation complex beta subunit beta-ketothiolase (FadA). The enzyme, FadA is involved in intermediary metabolism. The nucleotides FadA are 7672 to 8880 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 104.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the multifunctional fatty acid oxidation complex alpha subunit (FadB). The enzyme, FadB, is involved in intermediary metabolism. The nucleotides encoding FadB are 8855 to 11029 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 105.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding the M. leprae enzyme indolepyruvate decarboxylase (DCIP). The enzyme, DCIP, is involved in intermediary metabolism. The nucleotides encoding DCIP are located at positions 4228 to 2522 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 106.
One non-naturally occurring nucleic acid of the present invention has a sequence which corresponds to a sequence encoding a putative calcium binding protein. The protein is involved in intermediary metabolism. The nucleotides encoding this protein are 37098 to 36712 of Seq. ID No. 26. The putative amino acid sequence is set forth in Seq. ID No. 107.
Non-naturally occurring nucleic acids having a sequence of twenty or more nucleotides which correspond to a sequence of Mycobacterium leprae are useful as probes to identify Mycobacterium leprae genes or homologous genes in other mycobacteria or other related bacterial species. Nucleic acid sequences that are specific to Mycobacterium leprae genes include nucleotides at positions 4745 to 5074, nucleotides 35805 to 34525, nucleotides 16602 to 17078, nucleotides 36372 to 37181, nucleotides 6871 to 6071, nucleotides 20856 to 20293, nucleotides 21696 to 20893, nucleotides 22673 to 21702, nucleotides 25445 to 24921, and nucleotides 34517 to 34029 of Seq. ID No. 26. The putative amino acid sequences are set forth in Seq. ID Nos. 108-117.
One embodiment of the present invention features a non-naturally occurring protein or peptide of Mycobacterium tuberculosis. The protein or peptide preferably corresponds to a sequence encoded by Seq. ID No. 1. Preferably the protein or peptide is an antigen, or is involved in antibiotic or cell wall synthesis, intermediary metabolism, transport processes, nucleic acid biosynthesis or modification, cell division or antibiotic/drug resistance. Proteins and peptides of Mycobacterium tuberculosis are exemplified by Seq. ID Nos. 2-22, 118 and 119.
A further embodiment features a non-naturally occurring protein or peptide of Mycobacterium leprae. The protein or peptide preferably corresponds to a sequence within Seq. ID Nos. 23-26 or 120-140. Preferably, the protein or peptide is an antigen, or a protein involved in antibiotic or cell wall synthesis, intermediary metabolism, transport processes, nucleic acid biosynthesis or modification, cell division or antibiotic/drug resistance. Proteins and peptides of Mycobacterium leprae are exemplified by Seq. ID Nos. 27-117 and 141-411.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 120 are summarized in Table VI set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptide within Seq. ID No. 121 are summarized in Table VII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 122 are summarized in Table VIII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 123 are summarized in Table IX set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 124 are summarized in Table X set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 125 are summarized in Table XI set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 126 are summarized in Table XII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 127 are summarized in Table XIII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 128 are summarized in Table XIV set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 129 are summarized in Table XV set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 130 are summarized in Table XVI set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 131 are summarized in Table XVII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 132 are summarized in Table XVIII set forth below.
Mycobacterium leprae peptides, the function of such peptides, and the nucleotide positions associated with such peptides within Seq. ID No. 133 are summarized in Table XIX set forth below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 134 are summarized in Table XX below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 135 are summarized in Table XXI below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 136 are summarized in Table XXII below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 137 are summarized in Table XXIII below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 138 are summarized in Table XXIV below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 139 are summarized in Table XXV below.
Mycobacterium Leprae peptides, the functions of such peptides, and nucleotide positions associated with such peptides within Seq. ID No. 140 are summarized in Table XXVI below.
Proteins and peptides of the present invention have further utility for use as vaccines or as screens for new tuberculosis drugs. The purified proteins derived from Mycobacterium tuberculosis or leprae may elicit a specific immune response. The production of purified proteins by recombinant means is not limited to the use of pathogenic bacteria. The proteins of this invention, derived from M. tuberculosis or M. leprae, may also be expressed in M. bovis-BCG by recombinant means, and the resulting recombinant BCG cells may be used as a vaccine. See published PCT application WO90/00594, for example.
The availability of large quantities of Mycobacterium tuberculosis and/or leprae proteins and peptides allows the use of such proteins and peptides as specific binding agents for drugs. The proteins and peptides may also be used to elicit immune responses for the development of antibodies. Such antibodies may have therapeutic value. Proteins and peptides may also be used diagnostically to detect hypersensitivity reactions of individuals exposed to Mycobacterium tuberculosis or leprae. The proteins and peptides may be affixed to solid supports to detect antibodies from patient""s sera typical of hypersensitivity reactions.
For example, one method comprises the step of forming an admixture of a sera of an individual with a peptide corresponding to a peptide of Mycobacterium tuberculosis or leprae. Upon imposition of reaction conditions in the presence of an antibody to the peptide, an affinity complex forms. The method further includes the step of detecting the affinity complex which affinity complex is indicative of the presence of a hypersensitivity reaction.
One embodiment of the present invention features, as an article of manufacture, a kit comprising a non-naturally occurring nucleic acid corresponding to Mycobacterium tuberculosis or leprae nucleic acid. A further embodiment of the present invention features a kit comprising a non-naturally occurring peptide corresponding to a peptide of Mycobacterium tuberculosis or leprae or an antibody capable of binding such peptides.
These and other features will be apparent to individuals skilled in the art upon reading the detailed description of the present invention.